Landmark breakthroughs in the field of DNA synthesis, most importantly the Polymerase Chain Reaction (PCR) and the phosphoramidite chemistry, have lead to an increasing repertoire of modifications of natural DNA. In PCR, modified nucleoside triphosphates carrying a linker on the 5-position of pyrimidines or on the 7-position of 7-deazapurines can be incorporated using certain Family B polymerases [1]. The ease of incorporation of these building blocks into DNA strongly depends on the steric bulk and the molecular structure of the side chain. In principle all four naturally occurring nucleobases can be replaced by modified ones in a PCR [2]. If phosphoramidite chemistry is employed, in principle any molecular structure can be incorporated into DNA. The biggest limitation is that the modified nucleobases that are incorporated into DNA must be stable towards the conditions of phosphoramidite DNA synthesis and deprotection of the resulting DNA strand.
An approach circumventing both the problems of steric bulk as well as the potential chemical lability of the modifications has been developed in our group [3]. The modified nucleotides carry a linker containing an internal and a terminal alkyne. The internal alkyne facilitates incorporation via PCR as the steric bulk in close proximity to the nucleobase is minimized. The terminal alkyne is a reactive site for the Click reaction [4], which is a copper-catalysed Huisgen dipolar cycloaddition between azides and alkynes [5]. This reaction can be conveniently used for the postsynthetic labelling of biomolecules. Azides can thus be attached to DNA in a high-yielding reaction without encountering any significant side reactions, as natural biomolecules do not carry any azides or terminal alkynes. This approach is also described in PCT/EP2006/004017 the content of which is herein incorporated by reference.
According to previously described procedures only a single type of labelling group can be introduced in a site-selective manner. Thus it was an object of the present invention to overcome this limitation and allow the site-specific labelling of reporter molecules with at least two different labelling groups, e.g. dyes or other functional molecules in a consecutive way, thereby realizing an unprecedented versatility in modification.